1. Field of the Invention
In arriving at the gist underlying the concept of the instant invention, it was conceived that a new class of compounds of the general formula: ##STR2## wherein R can be either hydrogen or a second duplicate molecule (dimer), and R' can be any substituent, should be inhibitors of urease because of the positional proximity of the oxygen-nitrogen-sulfur functional groups in the molecule.
In the course of running comparative experiments to determine the necessity of the N-oxide functional group or the need for the dimeric, it was unexpectedly discovered that two other classes of compounds are also inhibitors of urease in soil systems, to wit, the pyridines and the pyrimidines. The pyridine class is of the general formula: ##STR3## wherein the ring can be any pyridine compound not substituted in the position alpha to the thiol group and R' can be any substituent.
The pyrimidine class is of the general formula: ##STR4## wherein the ring can be any substituted or unsubstituted pyrimidine compound, with R' being any substituent.
2. Description of the Prior Art
Compounds Which Inhibit Urease Hydrolysis of Urea: Most enzymes can be poisoned or inhibited by certain chemical reagents. From the study of enzyme inhibitors, valuable information can be obtained about substrate specificity of enzymes, the nature of the functional groups at the active site, and the mechanism of the catalytic activity.
There are two major types of enzyme inhibitors: irreversible and reversible. Irreversible inhibitors combine with or destroy a functional group of the enzyme molecule which is necessary for its catalytic activity. Reversible inhibitors generally are considered to be either competitive or noncompetitive. Competitive inhibitors compete with the substrate for binding to the active site but once bound cannot be transformed by the enzyme. An identifying feature of competitive inhibition is that it can be reversed by increasing the substrate concentration. Noncompetitive inhibitors do not bind at the site on the enzyme at which the substrate does; however, their binding to the enzyme alters the structure or conformation of the enzyme so that reversible inactivation of the catalytic site results.
The type of inhibition that is occurring can be determined by use of enzyme kinetics. However, such kinetic information on enzymes is limited to only a few clinically tested inhibitors, including the reversible inhibitors thiourea [G. B. Kistiakowsky, R. W. Shaw, "On the Mechanism of Inhibition of Urease," J. Am. Chem. Soc. 75, 866 (1953)]; phosphoramidate, fluoride ion, .beta.-mercaptoethanol [N. E. Dixon, R. L. Blakeley, B. Zerner, "Jack Bean Urease (EC 3.5.1.5), III. The Involvement of Active-Site Nickel Ion in Inhibition by .beta.-Mercaptoethanol, Phosphoramidate, and Fluoride," Can. J. Biochem. 58, 481-488 (1980)]; and hydroxamic acids such as acetohydroxamic acid.
A more empirical approach is to group the known inhibitors into classes according to their structures and how they are throught to interact with the urease enzyme active site. Gould [W. D. Gould, C. Hagedorn, R. G. L. McCready, "Urea Transformations and Fertilizer Efficiency in Soil," Advances in Agronomy 40, 209-238 (1986)] has recently suggested classifying inhibitors into three classes: (1) reagents which interact with the sulfhydryl groups (sulfhydryl reagents), (2) the hydroxamates, and (3) structural analogs of urea and related compounds. A special class of compounds can be added, a class containing agricultural crop protection chemicals which have been tested as urease inhibitors. These inhibitors have recently been reviewed in detail by Medina and Radel (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitor," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, in preparation at the time of this writing).
Sulfhydryl Reagents: Many organic and inorganic compounds, as well as metal ions, have been found to inhibit urease by reacting with the sulfhydryl groups in the enzyme. These may inactivate the enzyme by blocking an active-site group or causing a change in the tertiary structure of the enzyme.
A wide range of organic compounds has been tested as inhibitors (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitior," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, In Preparation). Most of the early work centered around the toluenes and quinones. Indeed, quite a number of the materials tested contain aromatic or unsaturated functional groups. Some of these materials are thought to be coordinators of nickel and are thus reversible, competitive inhibitors [R. L. Blakeley, B. Zerner, "Jack Bean Urease: the First Nickel Enzyme," J. Mol. Catal. 23, 263-292 (1984)].
The quinones inhibit urease activity by blocking essential groups at the active site, either by oxidation of the sulfhydryl group or by formation of addition products [R. Cecil, J. R. McPhee, "Sulfur Chemistry of Proteins," Adv. Protein Chem. 14, 255-389 (1959)] with the sulfhydryl group (equation 4). The heterocyclic mercapto compounds of Gould (Gould et al., 1978) appear to inhibit the enzyme by formation of a heterocyclic disulfide with the active-site sulfhydryl group (equations 4 and 5 infra). ##STR5##
Several inorganic materials have been tested as urease inhibitors (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitior," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, In Preparation). These materials generally are halide, carbonate, or sulfate salts. In addition, urea phosphate and nitric phosphate have been examined.
Fluoride is a competitive inhibitor which binds to the nickel cation in the active site [R. L. Blakeley, B. Zerner, "Jack Bean Urease: the First Nickel Enzyme," J. Mol. Catal, 23, 263-292 (1984)], and the behavior of the other halide probably is similar. It is likely that the acid inhibitors affect the optimum pH for urea hydrolysis rather than directly inhibit the enzyme.
A substantial number of transition metal compounds have been tested as urease inhibitors (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitior," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, In Preparation). The primary reaction for many of the metals is their reaction with the active sulfhydryl group. The mechanism of inhibition of C.sub.6 H.sub.5 Hg(OCOCH.sub.3) and most of the other cations is the reaction with the essential sulfhydryl group to form a metal sulfide complex (equation 6 infra). ##STR6##
An unusual group of compounds, the arylorganoboron compounds, was patented by Van Der Puy et al. (1984a). We suggest that rather than reacting with the essential sulfhydril group, that these compounds inhibit urease by reacting with the essential carboxylate of the enzyme active site, as shown in equation 7 infra: ##STR7##
Hydroxamic Acids and Hydroxamates: Dixon [N. E. Dixon, C. Gazzola, R. L. Blakeley, B. Zerner, "Jack Bean Urease (EC 3.5.1.5), A Metalloenzyme, A Simple Biological Role for Nickel?" J. Am. Chem. Soc. 97, 4131-4133 (1975)] has shown that hydroxamates inhibit urease by formation of a complex. These compounds are noncompetitive inhibitors [G. R. Gale, L. M. Atkins, "Inhibition of Urease by Hydroxamic Acids," Arch. Int. Pharmacodyn. Ther. 180, 289-298 (1969); K. Kobashi, J. Hase, K. Uehara, "Specific Inhibition of Urease by Hydroxamic Acids," Biochim. Biophys. Acta 65, 380-383 (1962)] which do not appear to be effective in soil systems [J. M. Bremner, L. A. Douglas, "Inhibition of Urease Activity in Soils," Soil Biol. Biochem. 3, 297-307 (1971); Gould et al. (1978)].
Structural Analogs of Urea: Until 1965, urease was thought to be specific to urea as a substrate. Since then, 11 substrates known to react with urease have been identified. As early as 1953 [G. B. Kistiakowsky, R. W. Shaw, "On the Mechanism of Inhibition of Urease," J. Am. Chem. Soc. 75, 866 (1953)], structural analogs of urea were found to inhibit urease. Thiourea, methylurea [G. B. Kistiakowsky, R. W. Shaw, "On the Mechanism of Inhibition of Urease," J. Am. Chem. Soc. 75, 866 (1953); Shaw, Raval, (1961)]; hydroxyurea [W. N. Fishbein, P. P. Carbone, "Urease Catalysis, II. Inhibition of the Enzyme by Hydroxyurea, Hydroxylamine, and Acetohydroxamic Acid," J. Biol. Chem. 240, 2407-2414 (1978)]; dihydroxyurea [W. N. Fishbein, "Urease Catalysis, III. Stoichiometry, Kinetics, and Inhibitory Properties of a Third Substrate: Dihydroxyurea," J. Biol. Chem. 244, 1188-1193 (1969)]; and various substituted phenyl ureas [S. Cervelli, P. Nannipieri, G. Giovanni, A. Perna, "Relations Between Substituted Urea Herbicides and Soil Urease Activity," Weed Res. 16, 365-368 (1976); S. Cervelli, P. Nannipieri, G. Giovanni, A. Perna, "Jack Bean Urease Inhibition by Substituted Ureas," Pestic. Biochem. Physiol. 5, 221-225 (1975)] were among the first. At least one of these, methylurea, has now been shown to act as a substrate rather than an inhibitor [P. V. Sundaram, K. J. Laidler, "Urease-Catalyzed Hydrolysis of Some Substituted Ureas and Esters of Carbamic Acid," Can. J. Biochem. 48, 1132-1140 (1970)] and hydroxyurea is simultaneously a substrate and an inhibitor [R. L. Blakeley, B. Zerner, "Jack Bean Urease: the First Nickel Enzyme," J. Mol. Catal. 23, 263-292 (1984)].
Recently, a great deal of interest has developed around the organophosphorus inhibitors, particularly the phosphoryl diamidates (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitor," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, in preparation at the time of this writing). Several of the more potent inhibitors come from this class of materials. In addition, it is likely that the first commercial inhibitor will come from this class of inhibitors. These inhibitors have the general structure shown below ##STR8## wherein R can be a wide variety of substituents and Z can be oxygen or sulfur.
Agricultural Crop Protection Chemicals: A large number of pesticides and herbicides also have been tested as urease inhibitors (R. M. Medina, R. J. Radel, "Mechanisms of Urease Inhibitior," in Ammonia Volatilization, D. E. Kissell, B. R. Bock, Editors, In Preparation). Although many of these materials inhibit urease, the inhibition effect generally is 30-60% as much as the more powerful phosphoryl diamidates. In several instances, there are conflicting reports indicating increasing, as well as decreasing, urea hydrolysis upon treatment with pesticide materials. It may be that the organophosphorus inhibitors possess crop protection activity as well.
Compounds Related to the Present Invention: Very little work has been reported in which substituted or unsubstituted pyridines have been tested as urease inhibitors. Pyridine-3-sulfonic acid (U.S. Pat. No. 3,547,614, Peterson et al., Dec. 15, 1970) (V), infra nicotinamide (VI), infra and nicotinohydroxamic acid (VII) [H. Junichi, K. Kyoichi, J. Biochem. (Toyoko) 62, 293-299 (1967)] have been shown to inhibit urease. Both Hydroxamic acids and sulfonic acids are known inhibitors of urease. ##STR9## These compounds inhibit the enzyme via these side chains and not through the pyridine moeity itself. Pyridoxine-HCL (VIII) infra is the only pyridine compound which may actually inhibit the enzyme [M. Rosetti, D. E. Mihelle, Farmacia 23, 141-146 (1975)]. ##STR10##
It may be possible that these new inhibitors and the phosphoroamides have substantially the same mechanism of inhibition, to wit, reacting with the essential sulfhydryl group(s) on the active site(s) of the urease. At this time, however, one can only speculate that the inhibitory properties of these three classes of inhibitors result either from some yet unidentified chemical properties and/or characteristics of the compounds themselves. If the mechanism is related to reacting with, or inhibiting of such sulfhydryl group(s), it might be classified as irreversible inhibition, but more probably as competitive inhibition.
Taking into consideration all of the information supra, one can establish that even though urease has been extensively studied for about 60 years, the mechanism of action and the mechanism of inhibition of this enzyme, especially in heterogeneous environments such as soils, are at best only partially known.